Conventional prenatal diagnostic methods with invasive procedures, such as chorionic villus sampling and amniocentesis, carry potential risks for both fetuses and mothers. Noninvasive screening of fetal aneuploidy using maternal serum markers and ultrasound are available but have limited sensitivity and specificity (Kagan, et al., Human Reproduction (2008) 23:1968-1975; Malone, et al., N Engl J Med (2005) 353:2001-2011).
Recent studies have demonstrated noninvasive detection of fetal aneuploidy by massively parallel sequencing of DNA molecules in the plasma of pregnant women is feasible. Fetal DNA has been detected and quantitated in maternal plasma and serum (Lo, et al., Lancet (1997) 350:485 487; Lo, et al., Am. J. hum. Genet. (1998) 62:768-775). Multiple fetal cell types occur in the maternal circulation, including fetal granulocytes, lymphocytes, nucleated red blood cells, and trophoblast cells (Pertl and Bianchi, Obstetrics and Gynecology (2001) 98:483-490). Fetal DNA can be detected in the serum at the seventh week of gestation, and increases with the term of the pregnancy. The fetal DNA present in the maternal serum and plasma is comparable to the concentration of DNA obtained from fetal cell isolation protocols.
Circulating fetal DNA has been used to determine the sex of the fetus (Lo, et al., Am. J. hum. Genet. (1998) 62:768-775). Also, fetal rhesus D genotype has been detected using fetal DNA. However, the diagnostic and clinical applications of circulating fetal DNA is limited to genes that are present in the fetus but not in the mother (Pertl and Bianchi, Obstetrics and Gynecology (2001) 98:483-490). Thus, a need still exists for a non invasive method that can determine the sequence of fetal DNA and provide definitive diagnosis of chromosomal abnormalities in a fetus.
The discovery of fetal cells and cell-free fetal nucleic acids in maternal blood in the past few decades and the application of high-throughput shotgun sequencing of maternal plasma cell-free DNA make it is available to detect small changes in the representation of chromosomes contributed by an aneuploid fetus in a maternal plasma sample. Non-invasive detection of trisomy 13, 18, and 21 pregnancies have been achieved.
However, as some studies show, GC bias introduced by amplification and sequencing placed a practical limit on the sensitivity of aneuploidy detection. GC bias might be introduced during the sample preparation and the sequencing process, under different conditions such as reagent composition, cluster density and temperature, which leads to differential sampling of DNA molecules with different GC composition and significant variation in sequencing data for chromosomes that are GC-rich or GC-poor.
To improve sensitivity, protocols for removal of the effect of GC-bias have been developed. Fan and Quake developed a method to computationally remove GC bias by applying weight to each GC density based on local genomic GC content, to ameliorate the number of reads mapped in each bin by multiplying corresponding weight (Fan and Quake PLoS ONE (2010) 5:e10439). However, the method has difficulty in dealing with sex chromosome disorders especially chromosome Y relevant disorders for the reason that the process may cause slight distortion of data which will interfere with the precision of detection.
Here, we describe a method to computationally remove the GC-bias in order to get a higher sensitivity in fetal genetic abnormality detection as well as avoid data distortion. This method defines parameters used for statistical test according to GC-content. In addition, we introduced the estimated fetal fraction into the diagnosis by binary hypothesis which showed higher sensitivity and specificity. Our method also shows it should be possible to increase the sensitivity of noninvasive detection of fetal genetic abnormality to preset precision for maternal sample containing a low fetal DNA fraction by sequencing more polynucleotide fragments. Resampling of maternal plasma in later gestational weeks may also increase the sensitivity of diagnosis.